The present invention relates generally to the field of diagnostic tests and, more particularly, to those tests useful in qualitative and quanitative determination of biological components, such as glucose and uric acid, in which tests such components are converted to an oxidizing substance, such as a peroxide.
Glucose oxidase enzymatically converts glucose to gluconic acid and hydrogen peroxide. The hydrogen peroxide thus formed can be reduced to H.sub.2 O by a peroxidatively active substance in the presence of an indicator system which is oxidized to produce a response, such as a color change. The chromogenic indicator o-tolidine has been used for some time in glucose test systems, but provides results which are subject to reduction of the oxidized indicator by interfering substances, such as ascorbic acid. Further, the safety of o-tolidine has been questioned.
Likewise, uricase enzymatically converts uric acid to allantoin and hydrogen peroxide. The hydrogen peroxide formed can be reduced to H.sub.2 O by a peroxidatively active substance in the presence of an indicator system, historically o-dianisidine.
More recently, Gochman and Schmitz have reported using 3-methyl-2-benzothiazolinone hydrazone hydrochloride with N,N-dimethylaniline to form an azo dye indicator in determinations of uric acid, Clin. Chem. 17:1154 (1971), and glucose Clin. Chem. 18:943 (1972). Even though it is asserted that the mixture with N,N-dimethylaniline was more resistant than o-tolidine, susceptibility to ascorbic acid interference produced significant error in reported uric acid and glucose concentrations.
The mechanism of oxidatively coupling heterocyclic hydrazones with phenols, aromatic amines and other compounds which can take part in the classic azo coupling reaction is reviewed briefly in Zollinger, Azo and Diazo Chemistry, Interscience, New York, p. 215-217 (1961). A summary of the original work, directed to the formation of azo dyes by oxidative coupling, of Hunig and co-workers in Germany (1957-68) is incorporated in Baer, Cationic Dyes for Synthetic Fibers, Venkataraman (ed.), The Chemistry of Synthetic Dyes, Vol. 4, Academic Press, N.Y., pgs. 188-193 (1971).
Hunziker, U.S. Pat. No. 3,979,262, adds a buffer, of citric or maleic acid, to the mixture of Gochman, supra, and discloses that, along with N,N-dimethylaniline, other aromatic amines can be used so long as they are not substituted in both the ortho and para positions. The buffer is also critical and maintains a predetermined pH range of from 3.2 to 4.7 for a uric acid determination and from 4.7 to 5.5 for a glucose or cholesterol determination.
The prior art, insofar as it teaches the use of hydrazone indicators in analysis for H.sub.2 O.sub.2, suggests that the reaction between MBTH and dimethylaniline is resistant to the effects of reducing substances in a sample. While this may be true relative to indicators such as o-tolidine, the use of such hydrazone indicators provides very poor indications in the presence of ascorbate.
Therefore, efforts by these prior workers have failed to provide an indicator system which is either substantially free of susceptibility to the effects of interfering substances or makes use of indicators recognized for their safety.